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Cardiovascular Research 2003 60(2):215-216; doi:10.1016/j.cardiores.2003.09.005
© 2003 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

PAF, PIP3 and NO: emerging role in reperfusion injury

Axel Gödecke*

Institut für Herz- und Kreislaufphysiologie, Heinrich-Heine-Universität, Universitätsstr.1, 40225 Düsseldorf, Germany

*Tel.: +49-211-8112675; fax: +49-211-8112675. Email address: Axel.Goedecke{at}uni-duesseldorf.de

Received 3 September 2003; Phosphatidylinositol 3-kinases (PI3Ks) constitute a ubiquitously expressed family of lipid kinases that phosphorylate the membrane lipid phosphatidylinositol 4,5-bisphosphate on position D3 of the inositol moiety, leading to formation of the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) [1]. PI3Ks are heterodimeric enzymes that are composed of a catalytic and a regulatory subunit. PI3K{alpha}, -β, and -{delta} (class Ia enzymes) respond to activated receptor tyrosine kinases like the insulin and PDGF receptors either by direct interaction of the regulatory subunit with phosphorylated receptor tyrosine residues or via adaptor molecules. In contrast, PI3K{gamma} (the only member of the class Ib family) is activated by β{gamma} subunits of heterotrimeric G proteins in response to stimulation of heptahelical G-protein-coupled receptors (GPCRs). In addition, activated RAS may exert a direct stimulatory effect on the catalytic subunit of PI3Ks. In response to receptor activation, cytosolic PI3Ks are recruited to the cell membrane, the site of their catalytic action. Membrane-localized PIP3, together with its primary metabolite PI-3,4P2 formed at the cell membrane, allows binding and activation of numerous signal transduction molecules predominantly via their plecstrin homology (PH) domain including protein kinase B (PKB/Akt), phosphoinositide-dependent kinase 1 (PDK1), tyrosine-kinases of the tek family, or GDP exchange factors for rac [2]. In line with the activation of these downstream targets, PI3Ks have been shown to regulate glucose metabolism, promote cell growth and proliferation, protect against apoptosis, and may mediate reorganization of the cytoskeleton, a process required for cell migration.

The activity of PI3Ks is counterbalanced by the tumor suppressor PTEN, a lipid phosphatase that removes the D3 phosphate moiety from PIP3. PTEN expression or activity has frequently been found to be decreased in advanced tumors, suggesting that constitutive PIP3 signaling may be part of tumor progression.

PI3Ks represent important second messenger-generating enzymes also in the cardiovascular system. A prominent example is the Ca2+-independent activation of endothelial NO synthase in endothelial cells via PKB/Akt-mediated phosphorylation [3], which may protect endothelial cells from apoptosis and induce endothelial migration [4]. In vascular smooth muscle cells, for example, the activity of L-type Ca2+ channels is stimulated via class Ia and Ib enzymes in response to tyrosine kinase and GPCRs [5].

Early reports demonstrated that in cardiac myocytes, PI3Ks were found to be involved in the hypertrophic response to a variety of stimuli including angiotensin II and {alpha}1-adrenoceptor stimulation. [6,7]. In addition, the cardiac activity of the PI3K{gamma} isoform is increased in response to pressure overload in vivo, suggesting a link between GPCR stimulation and hypertrophy. Downstream of PI3K, activation of the p70S6 kinase and the consequent increase in translational capacity of the ribosomes may be key events in the hypertrophic response. It was further demonstrated that in cardiac myocytes, PI3K signaling mediates the anti-apoptotic effect of insulin [8], most likely via Akt-mediated phosphorylation and inactivation of the pro-apoptotic protein BAD.

Given the important cellular functions of the PI3K family, the question arises how specific cellular functions are modulated by the distinct isoforms, which are frequently found to be coexpressed within the same cell. To examine the role of individual isoforms, transgenic mice with overexpression or deletion of single PI3K subunits or of the PTEN phosphatase represent valuable tools. Overexpression of a constitutively active or a dominant negative isoform of the catalytic p110{alpha} subunit in cardiac myocytes led to hypertrophy in the former and reduced heart size in the latter animals [9]. Genetic dissection of PI3K signaling was extended by genetic inactivation of PTEN and PI3K{gamma} in mice [10]. In PTEN knockouts, the enhanced levels of PIP3 led to cardiac hypertrophy, a finding that is in line with the expression of the constitutively active p110{alpha} subunit. In addition, PTEN-deficient hearts revealed a reduced contractile force, demonstrating that PI3K signaling is involved in regulating basal contractility. In contrast, PI3K{gamma}-deficient mice revealed an elevated contractility under basal conditions as well as under β2-adrenergic stimulation, an effect that could be attributed to the basal and induced levels of cAMP in cardiac myocytes. It is interesting to note that overexpression of the dominant negative p110{alpha} subunit has a clear effect on heart size but none on myocardial contractility. Thus, it remains to be elucidated by which mechanisms the {alpha} and {gamma} isoforms exert their specific effects.

In this issue of Cardiovascular Research, Alloatti et al. extend the analysis of the role of PI3K-mediated signal transduction in the regulation of myocardial contractile force in response to platelet activating factor (PAF). Using p110{gamma} knockout mice, the authors demonstrate that the negative inotropic effect of PAF in the heart depends on PI3K{gamma} in response to a Gi-mediated signal transduction pathway. Most interestingly, the negative inotropic effect of PAF could be blocked by NOS inhibition, suggesting that PKB/Akt-mediated phosphorylation and thus activation of eNOS is the critical event. Based on the earlier observation that PAF is released from the heart during reperfusion after global cardiac ischemia, Alloatti et al. further studied the relevance of PAF/PI3K{gamma} signaling for the outcome of ischemia–reperfusion in isolated hearts. It was clearly demonstrated that loss of PI3K{gamma} resulted in a significantly better post-ischemic recovery of contractile force during reperfusion than wild-type controls. However, post-ischemic function in wild-type hearts could be improved by both the PAF receptor antagonist WEB 2170 and the NOS inhibitor L-NMMA. Therefore, the work by Alloatti et al. identifies the PAF/PI3K{gamma}/NOS pathway as an important cause of myocardial dysfunction.

The functional consequences of a burst of NO and reactive oxygen species released during early reperfusion have been intensively analyzed during the past decade [11]. In this context, the data obtained by Alloatti et al. are fully compatible with earlier work, because pharmacological blockade of NOS in isolated rat hearts augmented functional post-ischemic recovery and this beneficial effect was attributed to the attenuated formation of peroxynitrite [12]. Similarly, Flögel et al. [13] demonstrated in isolated hearts of eNOS knockout mice as well as by pharmacological NOS inhibition that the recovery of cardiac function and energetics was improved after global cardiac ischemia. Thus, the work of Alloatti et al. adds another piece to the puzzle of signaling events determining the extent of reperfusion injury.

The involvement of PI3Ks in pathological processes as diverse as tumor progression, diabetes, allergies, and inflammation has raised considerable interest in pharmacological manipulation of PI3K activity [14]. Myocardial reperfusion injury also represents an important therapeutic target that now can be added to the list of disease states that might benefit from PI3K modulation. However, as also discussed by the authors, the net effect of PI3K{gamma} inhibition under in vivo conditions, i.e. in the blood-perfused heart after temporary coronary occlusion, is unclear. The cardiac actions of eNOS during reperfusion as assessed in the isolated heart appear to contribute to myocardial dysfunction in reperfusion. In contrast, other studies suggest that in vivo eNOS activation might play a protective role [15]. Possible mechanisms may be linked to reduced platelet aggregation [16] or leukocyte infiltration [15]. In addition, PI3K signaling in the heart appears to have an anti-apoptotic potential in response to tyrosine kinase activation, which might play a protective role in later stages after the onset of reperfusion. Thus, further analysis of the different PI3K isoform-specific knockout and overexpressing mouse models together with the development of isoform-specific inhibitors appears to be mandatory in order to decipher the therapeutic potential of PI3K modulation in the treatment of reperfusion injury.


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